Replicative senescence at the molecular level in vitro

Telomere-independent senescence (TIS)

Like p21, p16 is a CDK inhibitor involved in cell cycle arrest. Over expression of p16 causes G1 arrest in early passage cells by inhibiting the phosphorylation of the retinoblastoma protein (Kato et al, 1998). However, the removal of p16 activity results in only minimal lifespan extension that was terminated by senescence (Wei et al, 2003). p16 plays a role in maintaining senescence triggered by a short telomere. It appears that p21 is the initiating factor in the growth arrest observed in senescent cells, but p16 is required to maintain that state. p21 has been shown to initially increase in fibroblast cultures but later gradually decrease (Alcorta et al, 1996). During the decline of p21, p16 protein levels gradually increased in senescent cultures, reaching nearly 40-fold higher than in early cultures.

P16 is thought to trigger senescence independently of telomere length. For example, the inactivated p53 in two different fibroblast strains: WI-38 (from foetal lung) and BJ (from neonatal foreskin) resulted in the continuation of growth without Rb inactivation in BJ cells, whereas WI-38 cells fail to grow even when both Rb and p53 are inactivated. It was suggested that these two outcomes was due to the intrinsic differences in the ability to induce p16 at senescence. This proposal was based on the understanding that like WI-38, other human cells appear to undergo replicative senescence prior to telomere shortening by the induction of p16 (Itahana et al, 2003). The ability of p16 to cause growth arrest even after Rb inactivation also suggests that p16 can function independently of Rb activity. Further support for a telomere-independent mechanism was provided from studies which have shown that telomerase activity alone was insufficient to extend the replicative potential of human keratinocytes or mammary epithelial cells (Kiyono et al, 1998). It was shown that down-regulation of p16 in combination with telomerase activity did lead to replicative lifespan extension. Another study found that cyclin D1 over-expression in primary oral keratinocytes extend the lifespan, whereas the combination of cyclin D1 over-expression and p53 inactivation led to their immortalisation (Opitz et al, 2001). Cyclin D1 forms complexes with CDK4 which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). P16 inhibits cell cycle progression by inhibiting CDK4. It appears that the over-expression of cyclin D1 bypasses p16-TIS resulting in an extension of lifespan. In this instance the p16 mechanism has become redundant and the cells continue to divide until TDS mechanism is activated. This may be why these cells are immortalised by p53 inactivation.

This telomere-independent mechanism may be the primary trigger of cellular senescence in mice and rats. Mice and rats have telomeres which are 5-10 times longer than those of human cells (Shay and Wright, 2001). Despite this increase in length, rodent cells engineered to lack telomerase show telomere shortening with no effect on replicative potential. These cells senesce in the presence of long telomeres. Since mouse and rat cells repair DNA damage far less efficiently than do human cells, it has been suggested that such damage may be the trigger for p16 induction.

Replicative senescence at the molecular level in vitro

Telomere-dependent senescence (TDS)

Telomere attrition is thought to be the predominant mechanism which ultimately leads to a cell becoming senescent, caused by the inability of polymerase to completely replicate DNA at the 5’ end. The presence of a short telomere is thought to disrupt a protective telomeric structure, exposing loose ends of DNA which trigger a DNA damage response and consequently causes a cell to enter cellular senescence.

Human telomeres consist of short tandem GC-rich repeats (TTAGGG/AATCCC). These telomeric repeats are predominantly double-stranded with a 3’ single-stranded overhang terminating with a G-rich tail (Collins, 2000). The proposed mechanism by which telomeres and their associated proteins protect the chromosome ends is by creating large terminal loops known as a t-loop (Greider, 1999). In this structure, the double-stranded telomeric DNA is looped around and the 3’ single-strand overhang invades the duplex of telomeric repeats and forms a displacement loop (D-loop) structure is important for maintaining chromosomal stability. Without this protection, it is thought that the ends of chromosomes would become exposed, resembling double-strand breaks which can trigger cell-cycle arrest and senescence (Shore, 2001).

Telomeres can be elongated by adding telomeric repeats to the 3’ end of DNA. This can be carried out by telomerase, an enzyme which consists of an RNA molecule and a catalytic component known as hTERT. Telomerase is a reverse transcriptase which uses its RNA component as a template to reverse transcribes DNA back to the ends of chromosomes. Telomerase activity is repressed in most somatic tissue and reactivated in ~90% of human cancers (Artandi, 2006). Introduction of telomerase into normal somatic cells has been shown to extend replicative life-span (Bodnar et al, 1998) and not induce changes associated with a malignant phenotype (Jiang et al, 1999). Since telomerase is not present in somatic cells, they progressively get shorter.

The majority of short-telomere growth arrested cells is thought to occur in the G1 phase of the cell cycle, a process regulated by proteins such as p21. The appearance of DNA damage results in the activation of p53, a transcription factor whose activation results numerous downstream responses including the up-regulation of p21 and activation of DNA repair proteins. P21 is a cyclin dependent kinase (CDK) inhibitor which halts cell cycle progression by directly interacting and inhibiting cyclin-CDK complexes and consequently preventing the phosphorylation of Rb.

Experiments in which p21 was inactivated within senescent cells resulted in the ability of these cells to re-enter the S phase of the cell cycle, but were still unable to divide (Ma et al, 1999). It was concluded that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 is insufficient to permit cells to complete the cell cycle. Since p21 expression has been shown to be dependent upon p53, the impact of p53 inactivation on cell cycle progression was also investigated (Bond et al, 1999). Fibroblasts lacking p53 function bypassed replicative senescence and continued to proliferate for another 20 CPD before entering growth arrest. The point at which growth arrest occurs due to replicative senescence is also known as M1, whereas the second growth arrest point if replicative senescence is bypassed is known as M2 or crisis. Cells which enter crisis result in apoptosis rather than senescence. This suggests that when a cell becomes senescent at M1 it may not be due to the disruption of the telomere-end structure. Since there appears to be a reserved replicative capacity, it may only be at M2 that the telomere end-structure becomes disrupted, and this may be the reason why apoptosis is common at this point. The idea that a cell becomes senescent long before the telomere-end structure is disrupted suggests that telomere length may not be the trigger for replicative senescence and instead is due to another mechanism.

One such mechanism may be alterations or exposure of the telomere 3’ single-stranded overhang. One study reported that exposure of telomere 3’ overhang sequences induces senescence (Li et al, 2003). In this study, cells were exposed to oligonucleotides homologous to the telomere 3’ overhang sequence TTAGGG for 1 week. As a result, a senescent phenotype in cultured fibroblasts was observed. The team concluded that exposure the 3’ overhang by t-loop disruption or possibly DNA damage leads replicative senescence. Telomere shortening may result in the exposure of the 3’ overhangs, but the telomere end-structure is still maintained efficiently to prevent complete disruption. Interestingly, this 3’ overhang has been found to be eroded at replicative senescence indicating it as a possible trigger (Stewart et al, 2003). It was indicated that overhang erosion is the result of continuous cell division and not a consequence of senescence. Therefore it was concluded that this alteration in telomere structure and not overall telomere length serves as a trigger for replicative senescence.